Despite major advances in the subject of marijuana and cannabinoid (CB) research, there are only a few CB ligands currently available as FDA-approved drugs, partly because of the absence of experimental structures of CB receptors and a lack of knowledge of the conformational dynamics of the receptors, as well as a limited knowledge of the ligand-binding (orthosteric or allosteric) sites within the CB receptors. Very few allosteric modulators have been identified for CB receptors. There is great potential for discovery of novel CB ligands targeting either orthosteric or allosteric CB1 or CB2 receptor sites. Activators or inhibitors of these receptors are being investigated as therapeutic agents for many important human ailments including neuropathic pain, neuroinflammation, ischemic/reperfusion injury, anxiety, multiple sclerosis, and epilepsy. In this project, computational modeling of human CB1 and CB2 ligands, receptors, and receptor-ligand interactions will be carried out. We are interested in discovering orthosteric agonists selective for CB2 over CB1, peripherally- restricted antagonists, as well as allosteric CB modulators, all of which may be able to provide efficacy without causing negative psychotropic effects. Systematic, computationally-intensive research will yield novel active hit CB receptor modulators, which can later be developed into lead drug candidates. This will be accomplished through the following specific aims: Aim 1. To model, train and validate various ligand-based mathematical models to predict and classify CB ligands as agonists or antagonists. Literature- reported CB ligands and activity data as well as experimental data obtained for proprietary molecules being tested at the University of Mississippi, School of Pharmacy will be curated. Ligand-based models will be constructed, trained and validated. Aim 2. To construct, validate and simulate computational models of the CB1 and CB2 receptors in their active and inactive states. Receptor models will be developed, some that are tailored for agonists (active state) and some for antagonists or inverse agonists (basal or inactive states), utilizing structural information from multiple homologous GPCR templates. Aim 3. To apply integrated ligand-based and receptor-based virtual screening (VS) protocols to discover orthosteric and allosteric CB modulators. Validated models from Aims 1 and 2 will be used strategically in VS protocols to discover novel orthosteric and allosteric CB ligand chemotypes. We will screen repositories of small molecules including MarinLit, ZINC, and that of the National Center for Natural Products Research (NCNPR) and virtual libraries generated using computational tools to access extended chemical space. Top- ranked VS hits will be procured, purified if necessary, and characterized to confirm their structures. Aim 4. To conduct in vitro testing in CB1 and CB2 receptor binding and functional assays. Potential hit compounds from Aim 3 will be tested in CB1 and CB2 receptor binding and functional assays in the University of Mississippi School of Pharmacy NIH COBRE-supported In Vitro Research Core.